PROJECT SUMMARY: The technology for imaging neural activity in the rodent cortex has advanced dramatically. Using two-photon excited fluorescence microscopy and genetically encoded calcium indicators it is possible to record from many hundreds of neurons simultaneously in the cortex of awake, head-fixed mice during behavioral tasks. While cortical imaging is advancing rapidly, capabilities in other areas of the central nervous system remain underdeveloped. Neurons in the spinal cord play a critical role in coordinating rhythmic movements, such as walking and running. Because of its dense myelination, the white matter on the dorsal surface of the spinal cord is highly optically scattering and prevents imaging of underlying cells using two photon imaging. The goal of this grant is to develop tools that will overcome these limitations and enable imaging of neural activity in the spinal cord of mice that are awake and spine-fixed under a microscope while moving on a treadmill. These tools will enable studies that directly correlate patterns of neural activity in the spinal cord with limb movement and locomotor speed. These circuits have been studied primarily in reduced preparations, such as explanted neonatal spinal cords. The approach here would enable neural activity to be recorded in intact, adult animals that are awake and moving. In Aim 1, a treadmill that enables a mouse to be spine-fixed under the microscope will be developed. A long-term implantable spinal cord imaging chamber provides optical access to the spinal cord and enables the mouse to be spine-fixed on top of the treadmill. A non-invasive, markerless system for simultaneous gait and limb kinematic analysis will also be implemented. Care will be taken to optimize the setup so spine-fixed mice move with as normal a gait as possible. In order to image through the highly-scattering white matter, longer wavelength light and higher order nonlinear excitation of fluorescent labels is necessary. Using 1.3-m wavelength excitation pulses, green emitting fluorescent molecules can be three-photon excited. Similarly, 1.7-m light is suitable for three-photon excitation of red emitting fluorescent species. In Aim 2, the capabilities and limits of three-photon imaging in the spinal cord of mice will be explored. In preliminary data, cell structure and neural activity could be imaged as deep as 0.5 mm beneath the dorsal surface of the spinal cord, with even greater imaging depth likely achievable. The resolution, signal to noise, and depth penetration capabilities will be determined for each excitation wavelength and for both structural fluorescent labels and calcium indicators. In the final Aim, this new approach will be used to image the patterns of neural activity in a genetically-defined class of interneurons that are involved in locomotion. In particular, the activity of Chx10-expressing neurons will be correlated with limb movement and the hypothesis that the set of active cells changes with locomotion speed will be directly tested. Taken together, this project will establish the capability to image patterns of neural activity in the spinal cord of awake, behaving mice and demonstrate the utility of this approach in a study of one class of spinal cord interneurons.